My fermentation research has started to center on the behavior of a specific protein during cacao fermentation. Proteins constitute about 10-15% of the dry weight of cacao beans, and out of this amount 43% is the globular storage protein vicilin, while another 52% is the albumin fraction (Kumari et al., 2016). Voigt et al. (1994) showed that the breakdown of the vicilin protein is principally responsible for the cocoa-specific aroma precursors (the compounds which will form the aromas specific to cocoa and chocolate after they undergo Maillard reactions during roasting). Specifically, this protein is degraded cooperatively by two endogenous enzymes: an aspartic endoprotease and a carboxypeptidase (J. Voigt, Heinrichs, Voigt, & Biehl, 1994). First, the aspartic endoprotease breaks down the vicilin into small peptides less than 10 kDa in size (Guilloteau, Laloi, Michaux, Bucheli, & McCarthy, 2005) and then it is followed by the carboxypeptidase, which has a cleavage specificity for hydrophobic amino acids—notably leucine, phenylalanine, and alanine (J. Voigt, Heinrichs, et al., 1994). Later work confirmed the pH optima of the enzymes (3.5 for aspartic endoprotease and 5.5 for carboxypeptidase) that the original study had suggested (Jürgen Voigt, Textoris-Taube, & Wöstemeyer, 2018).

Briefly, what happens is, during a cacao fermentation, the beans start out covered in a wet, sugary, pulpy fruit. As fermentation progresses, that pulp is broken down and its sugars turn into alcohol and then later acid. As that acid diffuses into the bean, it causes elements inside the bean to rearrange. Now, some things that were sequestered are no longer separated from other components. So, the diffusion of liquids and rearrangement of components brings the enzymes (aspartic endoprotease and carboxypeptidase) and their substrate (vicilin) into contact with one another (Afoakwa, Paterson, Fowler, & Ryan, 2008). The acid that is brought in with those diffused liquids lowers the pH to the point that it reaches the pH optima of first one enzyme (the aspartic endoprotease, around 3.5). Then in a good fermentation the acid will volatilize or sometimes be neutralized by components produced by other organisms, and pH will rise again to the range of 5.5-6.0, into the optimal range of the carboxypeptidase.

So all of this is interesting, but there are still quite a few things we don’t know. For example, we don’t know the exact cleavage specificity of these enzymes for the vicilin globular protein. Also, we don’t know how this binding is affected by acidification; there is some evidence that the trimer conformation of the vicilin protein is disfavored under acidified conditions (Warwicker & Connor, 1995). We also don’t know if a specific mixture of organic acids might affect enzyme activity, or if different genetic varieties of cacao correspond with variations in the protein or enzymes. A lot remains to be studied.

What my research will focus on is modeling the specific mechanisms of this process. We plan to first study the conformational changes of vicilin under acidified conditions using circular dichroism (a method that measures changes in bond angles), then perform in vitro enzymatic digestion using vicilin and enzymes purified from raw cacao beans. Once we have identified the peptide products at each stage of the digestion, we can compare that with the circular dichroism data to create a 3D model of the likely binding sites at each stage in the enzymatic digestion. Ideally, this model should be predictive, allowing us to predict flavor precursors given certain input conditions.

More to follow another time on some interesting aspects of this protein and these two enzymes.

References

Afoakwa, E. O., Paterson, A., Fowler, M., & Ryan, A. (2008). Flavor formation and character in cocoa and chocolate: A critical review. Critical Reviews in Food Science and Nutrition, 48(9), 840–857. https://doi.org/10.1080/10408390701719272

Guilloteau, M., Laloi, M., Michaux, S., Bucheli, P., & McCarthy, J. (2005). Identification and characterization of the major aspartic proteinase activity in Theobroma cacao seeds. Journal of the Science of Food and Agriculture, 85(4), 549–562. https://doi.org/10.1002/jsfa.1777

Kumari, N., Kofi, K. J., Grimbs, S., D’Souza, R. N., Kuhnert, N., Vrancken, G., & Ullrich, M. S. (2016). Biochemical fate of vicilin storage protein during fermentation and drying of cocoa beans. Food Research International, 90, 53–65. https://doi.org/10.1016/j.foodres.2016.10.033

Voigt, J., Heinrichs, H., Voigt, G., & Biehl, B. (1994). Cocoa-specific aroma precursors are generated by proteolytic digestion of the vicilin-like globulin of cocoa seeds. Food Chemistry, 50(2), 177–184. https://doi.org/10.1016/0308-8146(94)90117-1

Voigt, J, Wrann, D., Heinrichs, H., & Biehl, B. (1994). The proteolytic formation of essential cocoaspecific aroma precursors depends on particular chemical structures of the vicilin-class globulin of the cocoa seeds lacking in the globular storage proteins of coconuts, hazelnuts and sunflower seeds. Food Chemistry, 51(2), 197–205. https://doi.org/10.1016/0308-8146(94)90257-7

Voigt, Jürgen, Textoris-Taube, K., & Wöstemeyer, J. (2018). pH-Dependency of the proteolytic formation of cocoa- and nutty-specific aroma precursors. Food Chemistry, 255, 209–215. https://doi.org/10.1016/j.foodchem.2018.02.045

Warwicker, J., & Connor, J. O. (1995). A model for vicilin solubility at mild acidic ph, based on homology modelling and electrostatics calculations. Protein Engineering, Design and Selection, 8(12), 1243–1251. https://doi.org/10.1093/protein/8.12.1243